Protein engineering is becoming a widely used tool in many areas of protein biochemistry. One engineering method is controlled protein ligation, and over the past ten years some progress has been made. For instance, synthetic-based chemistry allows the joining of synthetic peptides together through native chemical ligation and a 166 amino-acid polymer-modified erythropoiesis protein has been synthesized using this method. However, native chemical ligation relies on efficient preparation of synthetic peptide esters, which can be technically difficult to prepare for large polypeptides such as proteins. For example, the reaction sometimes is performed in an organic solvent to produce the requisite protein ester for ligation. Other ligation methods not requiring the production of protein esters generate protein thioesters. An intein-based protein ligation system was used to generate a protein by thiolysis of a corresponding protein-intein thioester fusion. A prerequisite for this intein-mediated ligation method is that the target protein is expressed as a correctly folded fusion with the intein, and that sufficient spacing between the target and intein is needed to allow formation of the intein-thioester. In many instances, the intein-fusion proteins can only be obtained from inclusion bodies when expressed in Escherichia coli, which often cannot be refolded. This difficulty significantly limits the application of intein-based protein ligation methods.
Purification of a tag-free recombinant protein often is challenging and often requires multiple chromatography steps. A tag can be linked to a recombinant protein, and after purification, the tag on the fusion may be cleaved from the target protein by treatment with an exogenously added site-specific protease. Additional chromatographic steps then are required to separate the target protein from the uncleaved fusion, the affinity tag, and the protease. For example, an N-terminal 6× His tag from a recombinant protein may be cleaved by an engineered 6× His-tagged aminoprotease, and a subtractive immobilized metal-ion affinity chromatography (IMAC) step can be used to recover the untagged target. Other methods may require two or more chromatography steps and even special treatment of the exogenous protease, such as biotinylation, to facilitate its removal.
Methods for the site-specific modification of proteins remain in high demand, and the transpeptidation reaction catalyzed by sortases has emerged as a general method for derivatizing proteins withvarious types of modifications. Target proteins are engineered to contain the sortase A recognition motif (LPXTG, SEQ ID NO: 45) near their C-termini. When incubated with synthetic peptides containing one or more N-terminal glycine residues and a recombinant sortase, these artificial sortase substrates undergo a transacylation reaction resulting in the exchange of residues C-terminal to the threonine residue with the synthetic oligoglycine peptide.